Cloning and Expression Analysis of COMT Gene in Pyrus bretschneideri cv. Dangshan Su Fruit

The development of pear stone cell is closely related to the synthesis,transfer and deposition of lignin. Caffeic acid o-methyltransferase( COMT) is a key enzyme in the lignin biosynthesis pathway that mainly catalyzes Syringyl lignin. In this study,a COMT gene named Pb COMT( Gen Bank Accession No. KC905086) from Pyrus bretschneideri cv. Dangshan Su fruit using RT-PCR and RACE methods was cloned. The Pb COMT c DNA comprises 1371 bp,with an ORF of 1098 bp,which encodes a putative protein containing 365 amino acid residues. Phylogenetic analysis indicated that Pb COMT shared high similarity with other plant COMTs,and showed 94% homology with apples. Furthermore,a prokaryotic expression vector p ET-32a-Pb COMT was constructed and transformed into E. coli BL21( DE3). Then a fusion protein with molecular mass 60. 0 k D was produced. Fluorescence quantitative expression analysis showed that the change of Pb COMT expression level was similar to that of lignin content,and also had great relevance with stone cell development rules. The present study is to elucidate the molecular mechanism of stone cell development in pear,which will provide a basis for further researches on the regulation of stone cell content and the improvement of pear taste.